Project Summary. The vast majority of breast cancer-related deaths are not caused by the primary tumor, but rather by metastases. Current studies mainly focus on the prevention of the early stages of metastasis. However, since tumor cells have likely left the primary tumor at diagnosis, inhibiting early stages of metastasis may not be the most effective way to inhibit metastatic burden. Therefore, an emphasis on developing therapeutics against late-stage metastasis is essential for improving survival in breast cancer patients. To better understand this complex process, I am examining the role of the developmental transcription factor Six2 in promoting metastatic burden. Six2 is a member of the Six family of transcription factors that are not only critical for development, but also for tumor progression and metastasis. Six2 is unique because it specifically affects the later stages of breast cancer metastasis, demonstrating its potential as a therapeutic target to decrease metastatic burden. My studies thus far have revealed novel roles for Six2 in breast cancer. I have shown that Six2 upregulates the expression of numerous stem cell-related genes, including a master regulator of stemness, Sox2. Six2 also increases the mammary stem cell population, as well as tumor-initiation in vivo. My data demonstrate for the first time that cancer cells may hijack Six2 to perform its developmental stem cell roles out of context, and that this role of Six2 may contribute to its ability to induce metastatic outgrowth. In this F99/K00 proposal, I outline a strategy to complete my dissertation studies, as well as how I will prepare to move on to a postdoctoral position. In Aim 1, I will describe my current findings regarding the role of Six2 in regulating stem cell phenotypes to promote late-stage metastasis. In Aim 2, I will outline my research strategy to complete my predoctoral studies, which will use stage-specific in vitro/in vivo models in combination with a comprehensive bioinformatics-driven candidate gene approach to continue to examine the role of Six2 in the later stages of metastasis. This approach will lead to the discovery of additional downstream effectors that could be targeted therapeutically to inhibit metastatic burden. Lastly, Aim 3 outlines my plans for postdoctoral research, and how I will identify an ideal postdoctoral mentor who will provide additional training in in vivo microscopy, single-cell analysis approaches and in vitro/ex vivo tissue-specific microenvironment engineering to examine other aspects of late-stage metastasis including how the microenvironment regulates the activity of metastasis-initiating cells (MICs). In addition, I will seek out an environment in which translational research is encouraged, as well as my continued commitment to academics and teaching. My current and future training, along with my dedication to cancer biology education and mentoring, is well aligned with the goals of the National Cancer Institute of providing opportunities and resources that allow current scientists to conduct cutting-edge research, while also inspiring future generations of cancer researchers.